How do cytotoxic T lymphocytes work in vivo?

Microbial Pathogenesis 1987 ; 3 : 315-318

Mini-review How do cytotoxic T lymphocytes work in vivo? A . Mullbacher and G . L . Ada Department of Microbiology, John Curtin School of Medical Research, Australian National University, G .P.O . Box 334, ACT 2601, Australia

The question-how do cytotoxic T lymphocytes (CTL) kill target cells in vivo? has intrigued many scientists since the first demonstration of this phenomenon in vitro by Rosenau and Moon .' Interest in this question has been re-awakened by the recent demonstration that CTLs recognize processed antigen in a fashion somewhat similar to antigen recognition by helper T cells .' The original experimental approach was to study cytotoxic cells in allogeneic systems in vitro ; the biological relevance of this work, it was argued, was primarily in surveillance against neoplasia . The advent of the 51 Cr release technique made the cytotoxic T cell assay one of the simplest, most sensitive and reliable assays in immunology . 3 A more convincing case for a physiological role for CTLs could be made when it was found that such cells, produced during a virus infection, would lyse syngeneic virus-infected target cells .' -' Their role in the immunopathology of disease caused by LCM virus together with the accompanying reduction of viral titers in infected organs was assumed to be due to lysis of virus-infected cells in target organs . The outcome of CTL activity could vary . In the case of LCM murine infection, the lysis of infected cells in the central nervous system led to increased mortality ; in contrast, the lysis of virus-infected cells during murine ectromelia infection in the liver, thus preventing continuing cycles of viral replication, led to increased survival . However, no direct evidence for the lysis of cells in vivo by CTLs has so far been found, whether it be a viral cell transfer system or the remission of neoplasia . A major reason for this is that CTLs, in addition to their potential to lyse cells directly (as shown from in vitro studies), have other phenotypic characteristics, notably the synthesis and release of a variety of soluble factors which, it could be claimed, are responsible for the observed in vivo phenomena . At this time, the authors are not aware of any definitive study which unequivocally shows that CTLs kill target cells in vivo (involving cell-cell contact) and that such a property is crucial for reduction of viral titers and host survival and/or immunopathological manifestations . This article lists and discusses on the one hand those properties of CTLs which are generally taken to support a direct cytolytic role in vivo, and on the other hand those experimental findings which cast doubt on such a role . As this is a mini-review, all work in this general area will not be referred to; we apologise if we overlook particularly relevant findings of other groups . Evidence consistent with a cytolytic role of CTLs in vivo Although not yet completely understood, much information is now available on specific molecules, intracellular organelles and extracellular structures which are involved in the process of lysis of target cell by a CTL .' These include the T cell antigen receptor 0882-4010/87/050315+04 $03 .00/0

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complex, electron dense secretory granules, pore-forming proteins, etc . Furthermore, cells other than CTLs which lyse target cells in vitro such as NK cells share some of these morphological features . In addition, no other function other than cytolytic activity has been proposed for such structures, suggesting that these structures have a particular biological role in vivo . In this connection, it was generally believed$ that the killing process was very largely a function of the effector T cell, i .e ., the target cell was murdered and the pore-forming proteins and secretory granules were the murder weapons . However, recent evidence,' using a variant thymoma clone as target, suggests that the target cell actively participates in the killing event, i .e ., it is a case of enforced suicide . The bulk of circumstantial evidence of the mechanism of in vivo lytic activity by CTLs derives from experiments in which in vivo phenomena correlate with in vitro cytotoxic activity . (1) CTLs are generated in vivo in response to infection by a variety of viruses and some bacteria and parasites ; killing of target cells in vitro can be demonstrated by these cells within as short a time as 30 min after their removal from the infected host . 10 (2) Transfer of such primary immune CTLs or in vitro-generated CTLs can cause reductions in viral titers (e .g . in the murine liver in the case of ectromelia infection" or t2,13 ) the murine lung in the case of influenza virus which range from 1 .5 to 4-5 logs10 in amount . It is important to note that transfer of T lymphocytes which secrete soluble factors but do not have demonstrable cytotoxic activity do not reduce viral titers in target organs . 14 As mentioned earlier, a corollary to the virus titer is immunopathology during LCM infection where transfer of specific CTLs causes enhanced pathological effects ." (3) In transfer experiments, the strength of the in vitro cytolytic activity correlated favourably with the level of viral titer reduction or increase in disease .' 1,12 (4) Such transfer experiments exhibited identical specificity patterns as seen in the in vitro cytotoxicity assays, i .e ., influenza-specific CTLs only reduced viral titers in influenza-infected mice . This was particularly impressive in experiments involving double infection of mice with two antigenically-distinct sub-types of influenza virus . Transfer of CTLs specific for one sub-type only reduced titers of the appropriate subtype virus whereas transfer of cross-reactive influenza-specific CTLs reduced titers of both viruses ." (5) Beside this viral specificity, viral reduction in target organs only occurred if transferred cells and the recipient were class 1 MHC compatible . This was best demonstrated in the elegant experiments of Kees and Blanden," using ectromelia virus and a class 1 MHC mutant murine strain . A mutation affecting only three amino acids in one of the class 1 MHC antigens abrogated the ability of the ectromelia-specific CTLs (raised in the infected, wild-type mouse strain) to reduce virus titers and confer protection . (6) Experiments by several groups 18,19 showed that CTLs recognize virus-infected target cells within 1-1 .5 hours of exposure of the cell to the infecting virus, thus implicating early virus-encoded gene products as the antigens recognized by the CTLs . This supports a role for CTLs being effective in vivo prior to the production of viral progeny during an infectious cycle . (7) In a recent study on LCM-induced T-cell mediated hepatitis, 20 a number of in vivo observed phenomena including liver enzyme levels in serum, the specificity of the transferred cells required for the induction of the disease, genetic parameters and the kinetics of manifestation of the disease correlated with the observed in vitro cytotoxic activity . In addition to the findings quoted above concerning viral-immune CTLs, alloreactive



Cytotoxic T lymphocytes in vivo

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CTLs have been implicated in the rejection of homografts such as thyroid glands ." Transfer of in vitro active alloreactive CTLs can specifically induce or cause the rejection of established allografts . In contrast, cells of non-lytic phenotype but capable of releasing lymphokines when confronted with the same alloantigen were unable to cause the rejection of such grafts . Arguments against a direct cytotoxic role for CTLs in vivo (1) An initial reaction against such a role for CTLs in vivo was a lack of signs of substantial cell death in target organs containing high levels of CTL activity . Such cell death is in fact seen in LCM-infected mice and it can be argued that in infections such as ectromelia and influenza, the target organs are well supplied with scavenger mononuclear cells which, by rapid clearance of cell debris, would obscure overt histological manifestation of tissue destruction . Furthermore, a number of viruses including ectromelia cause a large amount of tissue destruction . It would be difficult to distinguish lesions caused by cytopathic virus from those caused by CTLs . (2) Lin and Askonas 13 studied the activities of two CTL clones specific for influenza virus . Only one clone released interferon upon exposure to an infected target cell in vitro and only this clone, upon transfer to infected mice, was able to reduce lung virus titers . This finding suggested that release of gamma interferon might be directly involved in reduction of lung virus titers . Furthermore, a number of lymphokines known to be released by activated T cells such as lymphotoxins and others could contribute to immunopathology . In agreement with this finding, Schiltknecht and Ada 12 found that whereas cyclosporin, a drug known to inhibit the release of lymphokines from T cells, did not prevent in vitro cytotoxic activity, it did prevent reduction of virus titers by transferred CTLs in mice treated continually with the drug . However, one possible explanation proposed by Schiltknecht and Ada is that a lack of gamma interferon prevented the up-regulation of class 1 MHC antigen in cells productively infected by the virus, so they escaped lysis by CTLs . In fact, in view of recent findings of viral peptide recognition by class 1 restricted T cells, 2 it could be proposed that newly synthesized MHC antigens by infected cells could be important in presentation of viral epitopes recognized by CTLs . It will be of interest to see if similar findings are made with other viral systems . (3) It now appears most likely that CTLs recognize viral peptides attached to MHC antigens . In vitro, target cells can be sensitized to lysis by CTLs by exposure to viral peptides . 2 If this happened in vivo, there would be a risk that 'innocent' bystander (i .e ., uninfected) cells would be subject to unnecessary destruction . We believe, as do others, Z3 that such peptides produced by infected cells would not reach the necessary concentration in vivo to effectively sensitize adjacent uninfected cells . (4) Engers et al . 24 found that when a number of clones of CTLs, all of which demonstrated in vitro anti-P815 cytotoxic activity, were tested for their ability to eliminate P81 5 cells in vivo, only some were effective . This also argued that a direct correlation between in vitro and in vivo cytotoxic activity was questionable . There could be other explanations for these findings ; clones might vary in homing ability or expression of antigens so that some might be attacked by the host before their antiP81 5 activity was effective. (5) Immunization of animals with long term accepted allografts with small numbers of cells of reticuloendothelial origin syngeneic with the graft initiate the rejection of the graft . However, examination of secondary lymphoid tissues in these animals fails to show the presence of specific CTLs . An explanation for this finding could be the selective recruitment of CTLs to the site of the graft, or the possibility that graft



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rejection is mediated by such a small number of CTLs that they would be undetectable in an in vitro assay . Conclusion We believe that the arguments presented in this brief statement are entirely consistent with the original concept that CTLs act in vivo as they do in vitro, i .e ., by direct target cell lysis . Data and arguments in the past few years which have ostensibly been seen to argue against this proposal, have explanations which are at least as if not more tenable as the contrary argument . The topic will probably remain the subject of controversy until one group conducts an experiment which allows of a single explanation . As with so many other biological disciplines, finding an incontrovertible single explanation for an in vivo experiment is a difficult task . References 1 . Rosenau W, Moon HD . Lysis of homologous cells by sensitized lymphocytes in tissue culture . J Natl Cancer Inst 1961 ; 27 : 471-7 . 2 . Townsend ARM, Rothbard J, Gotch FM, Bahadur G, Wraith D, McMichael AJ . The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides. Cell 1986; 44 : 959-68 . 3 . Cerrottini JC, Brunner KT . Cell mediated cytotoxicity, allograft rejection and tumour immunity . Adv Immunol 1974 ; 18: 67-132 . 4 . Cole GA, Prendergast RA, Henney CS . In vitro correlates of lymphocytic choriomeningitis (LCM) virusinduced immune responses . Fed Proc 1973 ; 964 . 5 . Gardner ID, Bowern NA, Blanden RV. Cell-mediated cytotoxicity against ectromelia virus-infected target cells 1 Specificity and kinetics . Eur J Immunol 1974 ; 4 : 63-7 . 6 Leclerc JC, Gomard E, Plata F, Levy JP . Cell-mediated immune reaction against tumors induced by oncornaviruses. II . Nature of the effector cells in tumor cell cytolysis . Int J Cancer 1973 ; 11 : 426-32 . 7 . Young JD-E, Cohn ZA . Cell-mediated killing : a common mechanism? Cell 1986 ; 46 : 641-2 . 8 . Golstein P. Cytolytic T-cell melodrama . Nature 1987; 327 : 12 . 9 Ucker DS . Cytotoxic T lymphocytes and glucocorticoids activate an endogenous suicide process in target cells . Nature 1987 ; 327 : 62-4 . 10 . Blanden RV, Pang TE, Dunlop MBC . T cell recognition of virus-infected cells . In : Poste G, Nicolson GL, eds . Virus infection and the cell surface . Amsterdam : North Holland Publ . 1977 ; 249-90 . 11 . Blanden RV, Gardner ID . The cell-mediated immune response to ectromelia virus infection . I . Kinetics and characteristics of the primary effector T cell response in vivo . Cell Immunol 1976 ; 22 : 271-82 . 12 . Yap KL, Ada GL, McKenzie IFC . Transfer of specific cytotoxic T lymphocytes protects mice inoculated with influenza virus. Nature 1978 ; 273 : 238-9 . 13 . Lin YL, Askonas BA. Biological properties of an influenza A virus specific T-killer cell clone; inhibition of virus replication in vivo and induction of delayed-type hypersensitivity reactions . J Exp Med 1981 ; 154 :225-34 . 14 . Leung K-N, Ada GL . Different functions and immunopathological effects of subsets of effector T cells in murine influenza virus infection . Cell Immunol 1982 ; 67 : 312-24. 15 . Doherty PC, Zinkernagel RM . T cell-mediated immunopathology in viral infections . Transplant Rev 1974 ;19 :89-120, 16 . Lukacher AE, Braciale VL, Braciale TJ . In vivo effector function of influenza virus-specific cytotoxic T lymphocyte clones is highly specific . J Exp Med 1984; 160 : 814-26 . 17 . Kees U, Blanden RV . A single genetic element in H-2K affects mouse T cell antiviral function in pox virus infection . J Exp Med 1976; 143 : 450-5 . 18 . Jackson DC, Ada GL, Tha Hla R . Cytotoxic T cells recognize very early, minor changes in ectromelia virus-infected target cells . Aust J Exp Biol Med Sci 1976 ; 54 : 349-63 . 19 . Zinkernagel RM, Althage A . Antiviral protection by virus-immune cytotoxic T cells : infected target cells are lysed before virus progeny is assembled . J Exp Med 1977 ; 145 : 644-51 . 20 . Zinkernagel RM, Haenseler E, Leist T, Cerny A, Hengartner H, Althage A . T-cell mediated hepatitis in mice infected with lymphocytic choriomeningitis virus . J Exp Med 1986 ; 164 : 1075-92 . 21 . Lafferty KJ, Prowse SJ, Simeonovic C, Warren HS . Immunobiology of tissue transplantation : a return to the passenger leucocyte concept . Ann Rev Immunol 1983 ; 1 : 143-74 . 22 . Schiltknecht E, Ada GL . Influenza virus-specific T cells fail to reduce lung virus titres in cyclosporintreated, infected mice . Scand J Immunol 1985 ; 22 : 227-39 . 23 . Bevan MJ . Class discrimination in the world of immunology . Nature 1987 ; 325 : 192-3 . 24 . Engers HD, Glasebrook AL, Sorenson GD . Allogeneic tumor rejection induced by the intravenous injection of Lyt 2' cytolytic T lymphocyte clones . J Exp Med 1982 ; 156 : 1280-5 .